Fig 1.
Proximity-ligation assay (PLA) schematic and lectin specificity.
Biotinylated lectins with indicated specificities were added to first label specific sugars. Then, anti-ICAM-1 antibody and avidin, conjugated to complementary oligos, were added. When lectin-recognized sugar epitopes are less than 40 nm from anti-ICAM-1, the complementary oligos hybridize and amplify, producing red fluorescent puncta. Right panel shows N-glycan structures recognized by different lectins used (adapted from [19]. Man = Mannose; Gal = Galactose; GlcNac = N-acetylglucosamine; Sa = Sialic acid; R = varying N-glycan structures.
Table 1.
Atherosclerosis patient demographics (N.D. = no data).
Fig 2.
HM epitopes co-localize with ICAM-1 in high fat-induced mouse atherosclerosis.
Total, HM / hybrid, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 were measured in the innominate and left carotid arteries from ApoE-/- mice fed a normal or high fat diet. A) Shown are representative images of innominate arteries from paired lesion and non-lesion areas of the same vessel section. Red staining represents total ICAM-1, red puncta represent positive PLA staining for specific ICAM-1 N-glycoforms (indicated by arrows), and blue staining represents DAPI. # indicates the lumen of each vessel. Panels B and C show PLA staining of lesion areas when the anti-ICAM-1 antibody or avidin were excluded. Panel D shows total ICAM-1 staining in lesion versus non-lesion areas. *p<0.05 by t-test. Panel E shows number of puncta for HM / hybrid, HM, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 in lesion versus non-lesion areas. Data are mean ± SEM, each symbol represents a distinct mouse, n = 6. *p<0.05 compared to non-lesion via paired t-test. F. Representative images of CD68 staining (red) in lesion vs. non-lesion areas. G. Quantitation of CD68 staining in lesion and non-lesion areas. Data are mean ± SEM, each symbol represents a distinct mouse, n = 6. * = p<0.05 compared to non-lesion via paired t-test. ● from innominate arteries and ○ from left carotid arteries.
Fig 3.
HM / hybrid, HM, and α-2,6-sialylated ICAM-1 are increased in mouse atherosclerosis after induction of disturbed flow in vivo.
Panel A shows representative images of total ICAM-1 HM / hybrid, HM, α2,6-sialylated, and α2,3-sialylated ICAM-1 in the left carotid artery (after partial ligation) and paired right carotid artery (control). Positive PLA puncta are indicated by arrows. Panels B & C show total ICAM-1 staining in left versus right carotid artery and ICAM-1 N-glycoforms puncta, respectively. Data are mean ± SEM, each symbol represents an individual mouse, n = 3. *p<0.05 compared to RC via paired t-test. D. Representative images of CD68 staining (red) in LC vs. RC areas in mice. E. Quantitation of CD68 staining in LC and RC areas. Data are mean ± SEM, n = 3. *p<0.05 compared to RC via t-test. # indicates the lumen of each vessel.
Fig 4.
CD68 macrophage staining positively correlates with HM-ICAM-1 in animal models of atherosclerosis.
A-C. CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice fed HFD diet. n = 6 animals; n = 12 paired lesion and non-lesion vessel areas. D-F. CD68 staining (fluorescence) as a function of ConA, HHL, and SNA puncta, respectively, for ApoE-/- mice after partial carotid ligation n = 3 animals; n = 6 vessel samples. Best fit lines determined by linear regression with Pearson correlation analyses with indicated coefficients and p-values shown in each panel.
Fig 5.
HM / hybrid ICAM-1 is increased in human atherosclerosis.
Panel A shows representative images of total ICAM-1 (red staining) and specified N-glycoforms in human vessels with lesions spanning types 1–5. Red puncta represent positive PLA staining (as indicated by arrows). Panel B shows the quantification of total ICAM-1 in early (1–2) and late (3–5) disease stages. Each symbol represents a different patient, with same symbol representing multiple vessels from the same patient. Data are mean ± SEM, n = 7–10. * p<0.05 compared to types 1–2 via unpaired t-test. Panel C shows the quantification of HM / hybrid, HM, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 puncta in early (1–2) and late (3–5) disease stages. * = p<0.05 compared to early stage lesions by t-test. Data are mean ± SEM. n = 7–10. Panels D and E show staining of lesion areas when the anti-ICAM-1 antibody or avidin were excluded.
Fig 6.
CD68 macrophage staining positively correlates with HM / hybrid ICAM-1 in human atherosclerosis.
Panels A and B show representative images and quantitation, respectively, of CD68 staining in advanced and early type lesions, as well as IgG control staining. Data are mean ± SEM, n = 7–10 * p<0.05 compared to types 1–2 via unpaired t-test. C-E. ConA, HHL, and SNA puncta plotted against CD68 staining. Data are from n = 17 vessel samples collected from 12 subjects; each symbol represents a different subject. Best fit lines were determined by linear regression analyses indicated coefficients determined by Pearson correlation analyses.
Fig 7.
HM / hybrid, HM, α-2,6-sialylated, ICAM-1 are increased CKD patients with failed arteriovenous fistulas.
Panel A shows total ICAM-1 (red staining) and specified N-glycoforms in vessels from CKD patients with failed or successful AVF creation. Red puncta represent positive PLA staining (as indicated by arrows). B) Quantification of total ICAM-1 signal in failed and successful AVF samples (n = 4 each). Error bars are mean ± SEM; * p<0.05 compared to successful AVF samples by t-test. C) Quantification of HM / hybrid, HM, α-2,6-sialylated, and α-2,3-sialylated ICAM-1 as puncta per total ICAM-1 signal. Error bars are mean ± SEM; * p<0.05 compared to successful AVF samples by unpaired t-test.
Table 2.
AVF patient demographics.